Anabolic-androgenic steroids (AAS), which activate the human androgen receptor (hAR), are used clinically to treat hypogonadism, impotence, and muscle wasting disorders. In addition, there is significant non-prescribed, non-supervised abuse of AAS by athletes. This illicit use leads to serious and life threatening health problems, some irreversible. Understanding the mechanisms of action (MOA) of AAS can lead to better prevention of abuse and the reversing of adverse effects. AAS have varying anabolic and androgenic properties, but the mechanism of action has not been defined. When abused, AAS are taken at supraphysiological levels to increase muscle mass. AAS at these high levels may exert their actions through interactions at hAR, but also via repression of the human glucocorticoid receptor (hGR). In order to better understand the poorly defined MOA of AAS, anti-glucocorticoid activity must be addressed. We hypothesize AAS have multiple mechanisms of action including activation of the hAR, repression of the hGR via competitive antagonism, and repression of the hGR via nuclear receptor (NR) coactivator squelching. Aim 1 of this study will address AAS interactions at the hAR and hGR. Affinity and binding kinetics of AAS will be determined using surface plasmon resonance (SPR). Aim 2 will utilize fluorescence microscopy to address AAS binding to fluorescently labeled hAR and hGR, activation, and rate of receptor transport to nucleus. Transactivation via steroid.promoters will be evaluated with a luciferase assay in Aim 3; Aim 3a will assess the receptors alone while Aim 3b will assess the contribution of NR coactivators to the MOA of AAS using excess coactivators as well as mutated receptors. A recent report from NIDA stated anabolic steroid abuse by professional athletes highlights the fact that we are facing a very damaging message that is becoming pervasive in our society-that bigger is better and being the best is more important than how you get there. Together, the data obtained from the proposed studies will increase our understanding of the MOA of AAS, allow for the development of better therapeutics, and improve our ability to prevent abuse of these drugs. [unreadable] [unreadable] [unreadable]